Huntington's disease (HD) is a progressive neurodegenerative disorder that is inherited in a dominant fashion and results from a mutation that expands the polymorphic trinucleotide (CAG) tract in the Huntingtin gene (HTT). The average CAG tract size in the general population is 17-26 repeats (wildtype allele), however, in HD patients the CAG tract has expanded to 36 repeats or more (mutant allele) (Huntington's Disease Collaborative Research Group 1993. Cell 72(6):971-83). The HTT gene encodes the HTT protein and the expanded CAG tract results in a pathological increase in the polyglutamine repeats near the N-terminal of the protein. Individuals carry two copies of the HTT gene and one mutant allele is sufficient to result in HD.
Selective reduction or elimination (“knockdown” or “silencing”) of a defective gene product is a therapeutic approach that may be useful in many genetic diseases or disorders, however the HTT protein appears to have a role during development of the nervous system and a protective role in cells. In mouse models, constitutive knockout of the HTT gene is lethal during embryonic development (Nasir et al 1995. Cell 81(5):811-23), while adult inactivation of the HTT gene leads to progressive cell death in the brain and the testes (Dragatsis et al 2000. Nat. Genet 26:300-306). Reduction of huntingtin expression from the wildtype allele may, therefore, have negative consequences.
Sequence-based methods, such as antisense oligonucleotide (ASO), RNAinterference (RNAi) technology, microRNA (miRNA) or small hairpin interference RNA (shRNA), may be used for precise targeting of genes or nucleic-acid gene products. Some antisense (ASO), siRNA and ribozyme molecules have been developed that reduce huntingtin expression in vitro or in mouse models, (Handley et al 2006. Clin. Sci. (Lond). 110:73-88; Denovan-Wright et al., 2006. Gen Ther. 13:525-131; Koutsilieri et al 2007. J. Neural Trans. Suppl. 72:43-49).
Allele specific silencing has been demonstrated in cells expressing mutant genes in dominant human diseases—for example, spinocerebellar ataxia (SCA) type 3, SCA6, sickle cell anemia, frontotemporal dementia, amyotrophic lateral sclerosis, Familial amyloidotic polyneuropathy (FAP), Alzheimer's disease, slow channel congenital myasthenic syndrome and inherited dystonia.
The trinucleotide expansion that causes neurodegenerative disease, such as the CAG expansion in HTT that causes HD, is a clear allelic difference between the normal and mutated gene for these disorders (Bonini et al 2005. Neuron 48:715-718). However, the size of the disease-causing expansion mutation (in nucleotides) is frequently greater than the targeting capacity of siRNA or ASO molecules, and further, represents a highly repetitive element. This size may make the trinucleotide expansion difficult to target with siRNA or ASO methodologies.
PCT Publication WO 2008/005562 to Aronin et al. discloses several SNPs found in the Huntingtin gene, and discloses some specific nucleic acid sequences that may target one of the identified SNPs.
In order to effect an allele-specific reduction of expression of the mutant allele in a dominant gene disorder, such as those involving a polyglutamine expansion, greater detailed knowledge of the polymorphisms specific to a particular group of subjects, or an individual subject is required.